In article <199709040024.TAA06903 at borcim.wustl.edu>, brett
<brett at BORCIM.WUSTL.EDU> writes
>>I've treated my RNA with RNase-free DNase I (following Sambrok et al.,
>>and Ausubel et al. procedures) at least 3 consecutive times and I still
>>get DNA-generated bands in my RT-PCR. Does anybody know a method to
>>absolutely remove DNA contamination from RNA preparations?
>>Yes, this is indeed a problem. Most people don't use sensitive DNA detection
>methods when checking their digestions as we have. In fact someone in this
>lab is testing various nuclease combinations to obtain the best conditions
>for
>cleaning up RNA samples. She isn't finished, but she tells me 3 sequential
>digestions using cocktails of DNase I *and* ds- and ss- Exonucleases from
>Epicentre drop the DNA titer 7 orders of magnitude by qcPCR, with good recovery
>of RNA biologic activity.
>
Check out Biotechniques, June 97pp 1128-1132. They found that for RT-
PCR, DNA was digested much more efficiciently using DNAse I and 1mM Mn
than DNAseI and 1mM Mg.
They suggest that with Mg, DNAse I cleaves each strand independantly and
during inactivation of DNAse at 90C, the partially oevrlapping fragments
denature but then reanneal and reassocaite in the RT step. During the Rt
step the RT elongates these fragments, reducing the numebr of nicks and
increasing the likelihood that these fragments will reanneal also in
PCR. They postulate that Mn based cleavage is a different mechanism with
enhanced DNAse activity leading to shorter DNA fragments generated.
Must be worth a try.
Duncan
--
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.comhttp://www.genesys.demon.co.uk